Method for manufacturing a chromatography carrier

Abstract

A method for easily manufacturing a chromatography carrier having a large dynamic binding capacity for an antibody or its fragment, difficulty in leakage of the proteinaceous ligand during isolation, and difficulty in causing aggregation between carriers. A method for manufacturing a chromatography carrier comprising the following ligand binding process, ligand binding carrier bed formation process, ligand binding carrier liquid washing process, and ligand binding carrier stirring washing process. (Ligand binding process) A process of binding a proteinaceous ligand to a solid carrier (Ligand-binding carrier bed formation process) A process of forming a ligand-binding carrier bed by filling a container with the ligand-binding carrier obtained in the above ligand binding process. (Ligand-binding carrier liquid washing process) A process of washing the ligand-binding carrier bed formed in the above ligand-binding carrier bed formation process by passing it through a washing solution one or more times. (Ligand-bound carrier stirring and washing process) A process of stirring and washing the ligand-bound carrier after the above-mentioned ligand-bound carrier liquid washing process one or more times in a washing solution.

Description

Technology Field The present invention relates to a method for manufacturing a chromatography carrier. Background Technology In recent years, in the field of biopharmaceuticals, represented by antibody drugs, expression technology for target substances such as proteins has advanced significantly, and consequently, there is a demand for improved productivity in purification processes using chromatography. As a method to improve productivity, it is possible to reduce the concentration of impurities mixed in pharmaceutical raw materials, such as host cell-derived proteins and deoxyribonucleic acid, as much as possible in a single purification step, and to reduce the number of purification steps or processes. Consequently, there is a growing demand for chromatography carriers capable of realizing this, and the demand for dynamic binding capacity for antibodies or their fragments is particularly high. As a method for manufacturing such chromatography carriers, a method is known in which a synthetic polymer-based solid carrier or a natural polymer-based solid carrier is obtained through, for example, polymerization, cross-linking, or functional group introduction reactions, and a proteinaceous ligand is attached thereto (Patent Document 1). Prior art literature Japanese Patent Publication No. 2017-37069, Publication No. WO2008 / 146906, Pamphlet No. WO2019 / 121296 The problem to be solved In addition, the carrier bound to the proteinaceous ligand obtained above has impurities generated in each manufacturing process, including unreacted proteinaceous ligands, attached to it. Therefore, in order to prevent unreacted proteinaceous ligands from leaking out and being incorporated into the antibody during antibody purification, the carrier is sometimes washed with stirring after the ligand is bound (Patent Documents 2 and 3). However, in this washing process, unreacted proteinaceous ligands are not sufficiently removed, and there have been cases where proteinaceous ligands leak out during isolation. Therefore, the inventors examined the cleaning performance of the ligand-bound carriers by performing liquid-through washing rather than stirring washing, and found that aggregation between carriers is prone to occur. The ease with which aggregation between carriers occurs can cause variations in performance between lots. The problem that the present invention aims to solve is to provide a method for easily manufacturing a chromatography carrier that has a large dynamic binding capacity for an antibody or its fragment, is resistant to leakage of the proteinaceous ligand during isolation, and is resistant to aggregation between carriers. means of solving the problem The inventors have discovered that by combining a proteinaceous ligand with a solid carrier and then performing a liquid washing and agitation washing in this order at least once, the dynamic binding capacity of the chromatography carrier to the antibody or its fragment is increased, as well as making it difficult for the carriers to aggregate and for the proteinaceous ligand to leak during isolation. That is, the present invention is as follows <1> inside <7> It is to provide. <1> A method for manufacturing a chromatography carrier comprising the following ligand binding process, a ligand binding carrier bed formation process, a ligand binding carrier liquid-passing washing process, and a ligand binding carrier stirring washing process (hereinafter also referred to as the method for manufacturing a chromatography carrier of the present invention). (Ligand binding process) A process of binding a proteinaceous ligand to a solid carrier (Ligand-binding carrier bed formation process) A process of forming a ligand-binding carrier bed by filling a container with the ligand-binding carrier obtained in the above ligand binding process. (Ligand-binding carrier liquid washing process) A process of washing the ligand-binding carrier bed formed in the above ligand-binding carrier bed formation process by passing it through a washing solution one or more times. (Ligand-bound carrier stirring and washing process) A process of stirring and washing the ligand-bound carrier after the above-mentioned ligand-bound carrier liquid washing process one or more times in a washing solution. <2> The number of times the above-mentioned ligand-binding carrier liquid washing process passes through the liquid is 2 to 5, <1> Method for preparing a chromatography carrier as described in <3> A solid carrier washing process is further provided, wherein the solid carrier washing process comprises the following solid carrier bed forming process and solid carrier liquid washing process, and the solid carrier washed in the solid carrier washing process is used as the solid carrier in the ligand binding process. <1> or <2> Method for preparing a chromatography carrier as described in (Solid Carrier Bed Formation Process) A process of forming a solid carrier bed by filling a solid carrier into a container. (Solid carrier liquid washing process) A process of washing the solid carrier bed formed in the solid carrier bed forming process one or more times by passing it through a washing solution. <4> A solid carrier washing process is further provided, wherein the solid carrier washing process comprises the following solid carrier bed forming process, solid carrier liquid washing process, and solid carrier stirring washing process, and the solid carrier washed in the solid carrier washing process is used as the solid carrier in the ligand binding process. <1> inside <3> A method for manufacturing a chromatography carrier as described in any of the above. (Solid Carrier Bed Formation Process) A process of forming a solid carrier bed by filling a solid carrier into a container. (Solid carrier liquid washing process) A process of washing the solid carrier bed formed in the solid carrier bed forming process one or more times by passing it through a washing solution. (Solid carrier stirring and washing process) A process of stirring and washing the solid carrier one or more times in a washing solution after the solid carrier liquid washing process described above. <5> The number of times the liquid is passed through the solid carrier liquid washing process is 2 to 5 times, <3> or <4> Method for preparing a chromatography carrier as described in <6> The sum of the number of times the solid carrier liquid washing process and the number of times the ligand-bound carrier liquid washing process are 2 to 8 times, <3> inside <5> A method for manufacturing a chromatography carrier as described in any of the above. <7> The above proteinaceous ligand is one or more ligands selected from protein A, protein G, protein L, and derivatives thereof, <1> inside <6> A method for manufacturing a chromatography carrier as described in any of the above. Effects of the invention According to the method for manufacturing a chromatography carrier of the present invention, a chromatography carrier having a large dynamic binding capacity for an antibody or its fragment, difficulty in leakage of the proteinaceous ligand during isolation, and difficulty in aggregation between carriers can be easily manufactured. Specific details for implementing the invention [Method for manufacturing a chromatography carrier] The method for manufacturing a chromatography carrier according to the present invention comprises the following ligand binding process, ligand binding carrier bed formation process, ligand binding carrier liquid washing process, and ligand binding carrier stirring washing process. (Ligand binding process) A process of binding a proteinaceous ligand to a solid carrier (Ligand-binding carrier bed formation process) A process of forming a ligand-binding carrier bed by filling a container with the ligand-binding carrier obtained in the above ligand binding process. (Ligand-binding carrier liquid washing process) A process of washing the ligand-binding carrier bed formed in the above ligand-binding carrier bed formation process by passing it through a washing solution one or more times. (Ligand-bound carrier stirring and washing process) A process of stirring and washing the ligand-bound carrier after the above-mentioned ligand-bound carrier liquid washing process one or more times in a washing solution. Here, solid carrier resemblance used in the ligand binding process is explained. As a solid carrier, it is preferable to have a functional group capable of binding a ligand (e.g., a functional group selected from the group consisting of a cyclic ether group, a carboxyl group, -C(=O)-OC(=O)-, succinateimideoxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group) within the molecule. When such a solid carrier is used, it becomes easier to obtain a proteinaceous ligand with particularly excellent low leakage properties. Examples of solid carriers include particulate solid carriers, monolithic solid carriers, plate-like solid carriers, film-like solid carriers, fibrous solid carriers, and chip-like solid carriers, but particulate solid carriers are preferred, and porous particulate solid carriers (hereinafter also simply referred to as "porous particles") are more preferred. As for the porous particles, porous particles containing a polymer are preferred. These porous particles may be natural polymer-based porous particles composed of polysaccharides such as agarose, dextran, and cellulose, or synthetic polymer-based porous particles; however, in order to increase dynamic binding capacity or improve the uniformity of particle size, synthetic polymer-based porous particles are preferred. In addition, the porous particles are preferably water-insoluble. The solid carrier may be a commercially available product or one manufactured according to ordinary methods. Here, the method for manufacturing the solid carrier is described. When obtaining porous particles as a solid carrier, the porous particles can be manufactured by a method including a process of dispersing a monomer composition in an aqueous medium and performing suspension polymerization (hereinafter also referred to as process P1). -Process P1- As a monomer composition used in process P1, it is preferable to contain a monomer containing a functional group. The functional group contained in this monomer is preferably capable of being utilized in additional chemical reactions (such as reactions with a crosslinking agent) and may be capable of binding a ligand. Examples of functional groups may be selected from the group consisting of cyclic ether groups, carboxyl groups, -C(=O)-OC(=O)-, succinate imide oxycarbonyl groups, formyl groups, hydroxyl groups, and isocyanate groups. Among these, cyclic ether groups are preferred. Here, as the “cyclic ether group,” a cyclic ether group having 3 to 7 atoms constituting the ring is preferred. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group may be cyclic ether groups represented by the following formulas (4) to (9), but a cyclic ether group represented by formula (4), (6), or (9) is preferred, and a cyclic ether group represented by formula (4) is more preferred. [In the formula, R 11 to R 14 Each represents an independent hydrogen atom or alkyl group, and * represents a bond hand. R 11 to R 14 The number of carbon atoms in the alkyl group represented by is preferably 1 to 4, and more preferably 1 or 2. The alkyl group may be straight-chain or branched-chain, and examples include methyl groups, ethyl groups, n-propyl groups, isopropyl groups, n-butyl groups, sec-butyl groups, tert-butyl groups, etc. In addition, R 11 to R 14 As for that, a hydrogen atom is preferred. As for the functional group-containing monomer, a monomer having a functional group capable of binding a ligand and a polymerizable unsaturated group is preferred. Examples of such monomers include, for instance, glycidyl (meth)acrylate, 3-oxyranylpropyl (meth)acrylate, 4-oxyranylbutyl (meth)acrylate, 5-oxyranylpentyl (meth)acrylate, 6-oxyranylhexyl (meth)acrylate, 7-oxyranylheptyl (meth)acrylate, 8-oxyranyloctyl (meth)acrylate, (3-methyloxyranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, (Meth)acrylate monomers having cyclic ether groups, such as α-(meth)acryl-ω-glycidylpolyethyleneglycol and tetrahydrofurfuryl (meth)acrylate; aromatic vinyl monomers having cyclic ether groups, such as (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, (vinylphenethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, and 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers having cyclic ether groups, such as allylglycidyl ether; (meth)acrylate monomers having isocyanate groups, such as isocyanatoethyl (meth)acrylate; In addition to unsaturated dicarboxylic acid anhydride monomers such as maleic anhydride, methyl maleic anhydride, and glutaconic anhydride, examples include (meth)acrylic acid, 3,4-epoxy-1-butene, 3,4-epoxy-3-methyl-1-butene, etc. These monomers may be used individually or in combination of two or more types. Among these monomers, (meth)acrylate-based monomers having a cyclic ether group are preferred, and glycidyl (meth)acrylate is particularly preferred. The total amount of monomers containing functional groups is preferably 35 parts by mass or more, more preferably 45 parts by mass or more, and particularly preferably 55 parts by mass or more, with respect to 100 parts by mass of the total amount of monomers used in process P1, and also preferably 99 parts by mass or less, more preferably 90 parts by mass or less, and particularly preferably 85 parts by mass or less, with respect to 100 parts by mass of the total amount of monomers used in process P1. In addition, the monomer composition used in process P1 may contain, in addition to the above-mentioned functional group-containing monomer, a monomer other than the functional group-containing monomer (hereinafter also referred to as other monomer). Other monomers include polymerizable monomers containing unsaturated groups that do not possess functional groups capable of binding ligands. Other monomers are broadly classified into non-crosslinkable monomers and crosslinkable monomers, and either one of these may be used or a combination thereof. Examples of the above non-crosslinkable monomers include (meth)acrylate-based non-crosslinkable monomers, (meth)acrylamide-based non-crosslinkable monomers, aromatic vinyl-based non-crosslinkable monomers, vinyl ketone-based non-crosslinkable monomers, (meth)acrylonitrile-based non-crosslinkable monomers, N-vinylamide-based non-crosslinkable monomers, etc. These may be used individually or in combination of two or more types. Among the non-crosslinkable monomers, (meth)acrylate-based non-crosslinkable monomers and aromatic vinyl-based non-crosslinkable monomers are preferred. As the above (meth)acrylate-based non-crosslinkable monomer, for example, methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono(meth)acrylate, trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, butanetriol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, Examples include pentaerythritol mono(meth)acrylate, dipentaerythritol mono(meth)acrylate, inositol mono(meth)acrylate, etc. These can be used individually or in combination of two or more types. In addition, examples of the above (meth)acrylamide-based non-crosslinkable monomers include (meth)acrylamide, dimethyl (meth)acrylamide, hydroxyethyl (meth)acrylamide, (meth)acryloylmorpholine, diacetone (meth)acrylamide, etc. These may be used individually or in combination of two or more types. In addition, the above aromatic vinyl-based non-crosslinkable monomers may include, for example, styrenes such as styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, 4-tert-butylstyrene; vinyl naphthalenes such as 1-vinylnaphthalene, 2-vinylnaphthalene, etc. These may be used individually or in combination of two or more types. In addition, examples of the above vinyl ketone-based non-crosslinkable monomers include ethyl vinyl ketone, propyl vinyl ketone, isopropyl vinyl ketone, etc. These can be used individually or in combination of two or more types. In addition, examples of the above (meth)acrylonitrile-based non-crosslinkable monomers include acrylonitrile and methacrylonitrile. These can be used individually or in combination of two or more types. In addition, examples of the above N-vinylamide-based non-crosslinkable monomers include N-vinylacetamide, N-vinylpropionamide, etc. These can be used individually or in combination of two or more types. The total amount of non-crosslinkable monomer used is preferably 0.01 parts by mass or more, more preferably 0.05 parts by mass or more, and particularly preferably 0.1 parts by mass or more, with respect to 100 parts by mass of the total amount of monomer used in process P1, and also preferably 30 parts by mass or less, more preferably 15 parts by mass or less, and particularly preferably 5 parts by mass or less, with respect to 100 parts by mass of the total amount of monomer used in process P1. In addition, examples of the above-mentioned crosslinkable monomers include (meth)acrylate-based crosslinkable monomers, aromatic vinyl-based crosslinkable monomers, and allyl-based crosslinkable monomers. These may be used individually or in combination of two or more types. Furthermore, as for the crosslinkable monomer, a crosslinkable monomer with 2 to 5 functional groups is preferred, and a crosslinkable monomer with 2 or 3 functional groups is more preferred. Among the crosslinkable monomers, (meth)acrylate-based crosslinkable monomers and aromatic vinyl-based crosslinkable monomers are preferred. As the above (meth)acrylate-based crosslinkable monomer, for example, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, Butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, Examples include mannitol penta(meth)acrylate. These can be used individually or in combination of two or more types. In addition, examples of the above aromatic vinyl-based crosslinkable monomers include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene, etc. These can be used individually or in combination of two or more types. In addition, examples of the above-mentioned allyl-based crosslinkable monomers include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, diallyl trimellitate, diallyl cyanurate, diallyl isocyanurate, diallyl isocyanurate, and diallyl isocyanurate. These may be used individually or in combination of two or more types. In addition, as crosslinkable monomers, examples include, in addition to those exemplified above, dehydration condensation products of amino alcohols such as diaminopropanol, trihydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene. The total amount of crosslinkable monomer used is preferably 1 part by mass or more, more preferably 5 parts by mass or more, and particularly preferably 10 parts by mass or more, with respect to 100 parts by mass of the total amount of monomer used in process P1, and also preferably 50 parts by mass or less, more preferably 40 parts by mass or less, and particularly preferably 30 parts by mass or less, with respect to 100 parts by mass of the total amount of monomer used in process P1. Examples of aqueous media used in process P1 include aqueous solutions of water-soluble polymers, and examples of water-soluble polymers include hydroxyethylcellulose, polyvinyl alcohol, carboxymethylcellulose, polyvinylpyrrolidone, starch, gelatin, etc. The total amount of water-based media used is typically 200 parts by mass or more and 7,000 parts by mass or less per 100 parts by mass of the total monomer amount. In addition, when water is used as a dispersion medium in an aqueous medium, dispersion stabilizers such as sodium carbonate, calcium carbonate, sodium sulfate, calcium phosphate, and sodium chloride may be used. In addition, specific methods of process P1 may include, for example, a method of dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porous agent, suspending it in an aqueous medium, and heating it to a predetermined temperature to polymerize; a method of dissolving a polymerization initiator in a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porous agent, and adding it to an aqueous medium heated to a predetermined temperature to polymerize; or a method of suspending a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porous agent in an aqueous medium, heating it to a predetermined temperature, adding a polymerization initiator, and polymerizing. Radical polymerization initiators are preferred as polymerization initiators. Examples of radical polymerization initiators include azo-based initiators, peroxide-based initiators, and redox-based initiators; specifically, examples include azobis-isobutyronitrile, azobis-isobutyronic acid methyl, azobis-2,4-dimethylvaleronitrile, benzoyl peroxide, di-tert-butyl peroxide, and benzoyl-dimethylaniline peroxide. The total amount of polymerization initiators used is typically 0.01 parts by mass or more and 10 parts by mass or less per 100 parts by mass of the total monomer amount. The above porosizing agent is used to manufacture porous particles and exists together with the monomer during polymerization within the droplet, serving as a non-polymerizing component to form pores. The porosizing agent is not particularly limited as long as it can be easily removed from the porous surface; examples include linear polymers soluble in various organic solvents or mixed monomers, and these may be used in combination. As the above porous agent, for example, aliphatic hydrocarbons such as hexane, heptane, octane, nonane, decane, and undecane; alicyclic hydrocarbons such as cyclopentane and cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, naphthalene, and ethylbenzene; halogenated hydrocarbons such as carbon tetrachloride, 1,2-dichloroethane, tetrachloroethane, and chlorobenzene; aliphatic alcohols such as butanol, pentanol, hexanol, heptanol, 4-methyl-2-pentanol, and 2-ethyl-1-hexanol; alicyclic alcohols such as cyclohexanol; aromatic alcohols such as 2-phenylethyl alcohol and benzyl alcohol; Examples include ketones such as diethyl ketone, methyl isobutyl ketone, diisobutyl ketone, acetophenone, 2-octanone, and cyclohexanone; ethers such as dibutyl ether, diisobutyl ether, anisole, and ethoxybenzene; esters such as isopentyl acetate, butyl acetate, 3-methoxybutyl acetate, and diethyl malonicate; and linear polymers such as homopolymers of non-crosslinkable vinyl monomers. The porous agent may be used alone or in a mixture of two or more types. The total amount of the above porous agent used is typically 40 parts by mass or more and 600 parts by mass or less per 100 parts by mass of the total monomer amount. In addition, various surfactants, including anionic surfactants such as alkyl sulfate esters, alkylaryl sulfate esters, alkyl phosphate esters, and fatty acid salts, may be used in process P1. Additionally, polymerization inhibitors such as nitrites such as sodium nitrite, iodide salts such as potassium iodide, tert-butylpyrocatechol, benzoquinone, picric acid, hydroquinone, copper chloride, and ferric chloride may be used. Additionally, polymerization aids such as dodecyl mercaptan may be used. In addition, the polymerization temperature of process P1 can be determined according to the polymerization initiator, but is typically about 2 to 100°C, and 50 to 100°C is preferred. In addition, the polymerization time is typically 5 minutes to 48 hours, preferably 10 minutes to 24 hours. -Process P2- In addition, prior to the solid carrier cleaning process, a process (hereinafter also referred to as process P2) may be performed in which the porous particles obtained in process P1 are reacted with at least one selected from a crosslinking agent and a hydrophilizing agent. When both a crosslinking agent and a hydrophilizing agent are used, a hydrophilization reaction may be performed after the crosslinking reaction, or a crosslinking reaction may be performed after the hydrophilization reaction. In addition, the crosslinking reaction and the hydrophilization reaction may be performed simultaneously. When a monomer containing a functional group is used as the monomer composition in process P1, the crosslinking reaction causes a crosslinking agent to be added to a portion of the functional groups within the polymer molecules of the porous particles, thereby introducing a partial structure derived from the crosslinking agent. As a result, the residues of the functional groups are crosslinked with each other through the partial structure derived from the crosslinking agent. In addition, when a monomer containing a functional group is used as the monomer composition in process P, a hydrophilizing agent is added to a portion of the functional groups within the polymer molecules of the porous particles through the hydrophilization reaction, and a partial structure derived from the said hydrophilizing agent is introduced. The crosslinking agent used in process P2 may be one that can introduce a crosslinked structure by reacting with a functional group capable of binding a ligand, but a crosslinking agent that can introduce a crosslinked structure by reacting with a functional group capable of binding a ligand and also contains at least two groups represented as -C(=O)-NH- within the molecule is preferred. In the case where the porous particles obtained in process P1 have cyclic ether groups, specifically, a crosslinking agent containing at least two groups represented as -C(=O)-NH-NH2 in the molecule as crosslinking groups, a crosslinking agent containing at least two groups represented as -C(=O)-NH- and at least two carboxyl groups in the molecule as crosslinking groups, etc. may be used. In the case where the porous particles obtained in process P1 have a carboxyl group, -C(=O)-OC(=O)-, succinate imideoxycarbonyl group, formyl group, or isocyanate group, specifically, a crosslinking agent such as one containing at least two groups represented as -C(=O)-NH-NH2 in the molecule as crosslinking groups may be used. As a crosslinking agent comprising at least two groups represented by -C(=O)-NH- as described above within the molecule, examples include dicarboxylic acid dihydrazides such as oxalyl dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, 2,3-dihydroxysuccinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, octaneic acid dihydrazide, nonaneic acid dihydrazide, sebacic acid dihydrazide, dodecaneic acid dihydrazide, phthalic acid dihydrazide, isophthalic acid dihydrazide, terephthalic acid dihydrazide, quinolinic acid dihydrazide; and tricarboxylic acid trihydrazides such as cyclohexane tricarboxylic acid trihydrazide; Examples include (alkylenebisimino)bis(oxoalkano) series such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide). A crosslinking agent may be used alone or in combination of two or more types. Among these crosslinking agents, dicarboxylic acid dihydrazides and (alkylenebisimino)bis(oxoalkano) series are preferred, and dicarboxylic acid dihydrazides are more preferred, in order to improve liquid permeability, pressure resistance characteristics during liquid passage, and antifouling properties. In addition, in process P2, a crosslinking agent other than a crosslinking agent containing at least two groups represented by -C(=O)-NH- within the molecule may be used. Examples of such crosslinking agents include polyfunctional isocyanate-based crosslinking agents, polyfunctional epoxy-based crosslinking agents, polyfunctional aldehyde-based crosslinking agents, polyfunctional thiol-based crosslinking agents, polyfunctional oxazoline-based crosslinking agents, polyfunctional aziridine-based crosslinking agents, metal chelate-based crosslinking agents, etc. The total amount of crosslinking agent used is preferably 0.01 molar equivalent or more and 0.8 molar equivalent or less per 1 mol of functional group derived from the functional group-containing monomer, more preferably 0.05 molar equivalent or more and 0.7 molar equivalent or less, and particularly preferably 0.1 molar equivalent or more and 0.6 molar equivalent or less. As a hydrophilizing agent used in process P2, in order to improve antifouling properties or low leakage of protein ligands, a compound having a total of at least two hydrophilic groups selected from hydroxyl groups and mercapto groups in the molecule is preferred, and a compound having a total of two to four hydrophilic groups selected from hydroxyl groups and mercapto groups in the molecule is more preferred. Examples include alcohols having mercapto groups in the molecule, such as mercaptoethanol and thioglycerol; and polyhydric alcohols such as glycerol and diglycerol. The hydrophilizing agent may be used as a single type or in combination of two or more types. Among these, alcohols having a mercapto group within the molecule are preferred to improve antifouling properties or low leakage of protein ligands, and thioglycerol is particularly preferred. The total amount of hydrophilic agent used is preferably 0.5 molar equivalents or more and 10 molar equivalents or less per 1 mole of functional group derived from the functional group-containing monomer, more preferably 1 molar equivalent or more and 8 molar equivalents or less, and particularly preferably 2 molar equivalents or more and 6 molar equivalents or less. Process P2 may be carried out in the presence of a basic catalyst. Examples of basic catalysts include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, diisopropylethylamine, etc., and one type may be used alone or in combination of two or more types. In addition, the reaction time of process P2 is not particularly limited, but is typically about 0.5 to 72 hours, and preferably about 0.5 to 48 hours. In addition, the reaction temperature can be appropriately selected below the boiling point of the solvent, but is typically about 2 to 100°C. -Process P3- In addition, when a ligand is bonded to a porous particle through a linker (spacer) in the ligand binding process, a process (hereinafter also referred to as process P3) may be performed prior to the solid carrier washing process, in which the porous particle obtained in process P1 or process P2 is reacted with a compound that provides a linker. Examples of compounds that provide linkers include, for instance, diglycidyl ethers of aliphatic polyhydroxy compounds such as ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, 1,2-propylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, and glycerol diglycidyl ether; and polyglycidyl ethers of aliphatic polyhydroxy compounds such as sorbitol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, diglycerol polyglycidyl ether, and polyglycerol polyglycidyl ether. Among these, when a hydrophilization reaction is performed in process P2, diglycidyl ethers of aliphatic polyhydroxy compounds are preferred. In order to increase reaction efficiency, it is preferable to carry out the linker introduction reaction in a buffer with a pH of 7 to 14. In addition, the reaction time of the linker introduction reaction is not particularly limited, but is typically about 0.5 to 72 hours. In addition, the reaction temperature can be appropriately selected below the boiling point of the solvent, but is typically about 2 to 100°C. (Solid carrier cleaning process) In order to increase the hydrophilicity of the surface of the solid carrier or to make it difficult for the protein ligand to leak during isolation, the method for manufacturing the chromatography carrier of the present invention further comprises a solid carrier washing process in addition to the ligand binding process, the ligand binding carrier bed forming process, the ligand binding carrier liquid washing process, and the ligand binding carrier stirring washing process, and it is preferable to use the solid carrier washed in the solid carrier washing process as the solid carrier in the ligand binding process. The solid carrier cleaning process is a process for cleaning the solid carrier. It is preferable that the solid carrier cleaning process be performed using a cleaning solution (hereinafter also referred to as "solid carrier cleaning solution"). As a cleaning solution for a solid carrier, an aqueous cleaning solution is preferred. "Aqueous cleaning solution" means a cleaning solution containing at least water. Examples of aqueous cleaning solutions include those containing water or a mixture of water and a lower alcohol. Examples of lower alcohols include one or more selected from ethanol and isopropanol. In addition, as a cleaning solution for a solid carrier, at least one cleaning solution selected from a cleaning solution containing hydrogen peroxide, a cleaning solution containing peracetic acid, and a cleaning solution with a pH of 0 or higher and 3 or higher and 12.5 or lower (excluding cleaning solutions containing hydrogen peroxide and peracetic acid) is preferred in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for protein ligands to leak during isolation, to increase dynamic binding capacity, and to suppress aggregation between carriers. When such a cleaning solution is used, the surface of the solid carrier becomes hydrophilic, and the dynamic binding capacity of the obtained chromatography carrier for antibodies or their fragments, low leakage of protein ligands, and low aggregation are improved. Furthermore, it becomes possible to use cleaning solutions of various pH values ​​as cleaning solutions for the ligand-binding carrier liquid-passing cleaning process or the ligand-binding carrier stirring cleaning process. For example, even when a cleaning solution with a pH greater than 3 and less than or equal to 12.5 is used in a ligand-binding carrier liquid-washing process or a ligand-binding carrier stirring-washing process, a chromatography carrier is obtained in which it is difficult for proteinaceous ligands to leak out during isolation. Although the reason why it is difficult for ligands to leak out during isolation in this way is not necessarily clear, the inventors presume that the surface of the solid carrier becomes hydrophilic by the cleaning solution and thus possesses excellent antifouling properties, and thus unreacted proteinaceous ligands that are generally attached during the subsequent ligand binding process become easier to clean even if the pH of the cleaning solution is, for example, greater than 3 and less than or equal to 12.5. For example, the inventors presume that when the solid carrier contains functional groups capable of binding ligands, such as cyclic ether groups or hydroxyl groups, or functional groups such as carbonyl bonds within its molecule, the hydrophilicity increases due to the hydrolysis of these functional groups or the presence of large amounts of hydroxyl groups or carboxyl groups, thereby providing excellent antifouling properties. Furthermore, the method for manufacturing a chromatography carrier according to the present invention allows for the production of a chromatography carrier with a large dynamic binding capacity for antibodies or their fragments, even when a solid carrier washing process is performed prior to ligand binding, such that the ligand is sufficiently bound to the solid carrier during the ligand binding process. Although the reason for obtaining such a dynamic binding capacity is not necessarily clear, the inventors presume that one of the reasons is that damage to the chromatography carrier can be significantly reduced by performing multiple washes in an appropriate manner. In addition, in the present invention, "hydrophilization" means that the affinity for water is increased. As a cleaning solution for a solid carrier, a cleaning solution with a pH of 0 or higher and 3 or a pH greater than 12.5 and 14 or lower is preferred in order to increase the hydrophilicity of the solid carrier surface or to make it difficult for protein ligands to leak during isolation, and a cleaning solution with a pH greater than 12.5 and 14 or lower is more preferred. The pH of the cleaning solution containing hydrogen peroxide is preferably 4.5 or higher and less than 7, more preferably 5 or higher and less than 6.8, particularly preferably 5.5 or higher and less than 6.5, and most preferably 6 or higher and less than 6.5, in order to make it difficult for the protein ligand to leak when isolated. In addition, the concentration of hydrogen peroxide in the cleaning solution is preferably 0.001M or more and 10M or less, more preferably 0.005M or more and 5M or less, and particularly preferably 0.01M or more and 1M or less, in order to make it difficult for the protein ligand to leak during isolation. The pH of the washing solution containing peracetic acid is preferably 1 or more and less than 7, more preferably 2 or more and less than 6, particularly preferably 3 or more and less than 5, and most preferably 4 or more and less than 5, in order to make it difficult for the protein ligand to leak out during isolation. In addition, the concentration of peracetic acid in the washing solution is preferably 0.001M or more and 5M or less, more preferably 0.005M or more and 3M or less, and particularly preferably 0.01M or more and 1M or less, in order to make it difficult for the protein ligand to leak during isolation. In addition to a cleaning solution containing hydrogen peroxide and a cleaning solution containing peracetic acid, the solid carrier cleaning solution includes a cleaning solution with a pH of 0 or higher and 3 or lower, and a cleaning solution with a pH greater than 12.5 and less than or equal to 14. The pH of the cleaning solution is preferably 0 or higher and 2.5 or lower, more preferably 0 or higher and 2 or lower, in order to increase the hydrophilicity of the solid carrier surface or to make it difficult for the protein ligand to leak out during isolation. The pH of the cleaning solution, which is greater than 12.5 and less than or equal to 14, is preferably 12.7 or greater and less than or equal to 14, more preferably 13 or greater and less than or equal to 14, and particularly preferably 13.2 or greater and less than or equal to 14, in order to increase the hydrophilicity of the solid carrier surface or to make it difficult for the protein ligand to leak out during isolation. When the pH of the washing solution is set to 13.2 or higher and 14 or lower, the hydrophilicity of the solid surface of the chromatography carrier obtained is increased, and furthermore, it becomes particularly difficult for protein ligands to leak out during isolation. As for the solid carrier washing solution, it is preferable to include a strongly acidic pH adjuster or a strongly basic pH adjuster to increase the hydrophilicity of the solid carrier surface or to make it difficult for proteinaceous ligands to leak during isolation, and it is more preferable to include a strongly basic pH adjuster. As a strong acidic pH adjuster, inorganic acid-based strong acidic pH adjusters such as sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is more preferred. In addition, as a strong basic pH adjuster, alkali metal hydroxides such as lithium hydroxide, potassium hydroxide, and sodium hydroxide are preferred, and potassium hydroxide and sodium hydroxide are more preferred. When a strong basic pH adjuster is used, it becomes easier to adjust the pH of the cleaning solution to a range of greater than 12.5 and less than or equal to 14, and when a strong acidic pH adjuster is used, it becomes easier to adjust the pH of the cleaning solution to a range of greater than or equal to 0 and less than or equal to 5. In addition, the concentration of a strong acidic pH adjuster or a strong basic pH adjuster in the solid carrier washing solution is preferably 0.001M or more and 25M or less, more preferably 0.005M or more and 10M or less, even more preferably 0.01M or more and 5M or less, even more preferably 0.05M or more and 2.5M or less, even more preferably 0.25M or more and 1.5M or less, and particularly preferably 0.5M or more and 1M or less, in order to make it difficult for the protein ligand to leak during isolation. When the concentration of a strong acidic pH adjuster or a strong basic pH adjuster is 0.25 M or higher or 0.5 M or higher, the low leakage of the protein ligand becomes particularly excellent. The solid carrier washing solution may contain a buffer or two or more pH adjusters in order to adjust to a desired pH or to maintain a target pH. Washing using a solid carrier washing solution may be performed a total of once or a total of two or more times. However, in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for proteinaceous ligands to leak during isolation, and to suppress aggregation between carriers, it is preferably performed a total of two or more times, more preferably a total of 2 to 20 times, even more preferably a total of 2 to 15 times, even more preferably a total of 2 to 10 times, even more preferably a total of 2 to 8 times, and particularly preferably a total of 3 to 4 times. In addition, when washing a total of two or more times, the type of solid carrier washing solution used in each wash may be the same or different, but the same type is preferred. When the total number of washes using the solid carrier washing solution is three or more, the low leakage of protein ligands is particularly excellent. In addition, when the total number of washes using the solid carrier washing solution is four or fewer, the low aggregation is particularly excellent. The amount of solid carrier cleaning solution used per cleaning cycle is preferably 40 parts by volume or more, more preferably 60 parts by volume or more, and particularly preferably 80 parts by volume or more, with respect to 100 parts by volume of dry solid content of the solid carrier, and also preferably 1,000 parts by volume or less, more preferably 500 parts by volume or less, and particularly preferably 300 parts by volume or less, with respect to 100 parts by volume of dry solid content of the solid carrier. As for the specific range, with respect to 100 parts by volume of dry solid content of the solid carrier, it is preferable to have 40 parts by volume or more and 1,000 parts by volume or less, more preferable to have 60 parts by volume or more and 500 parts by volume or less, and particularly preferable to have 80 parts by volume or more and 300 parts by volume or less. The cleaning temperature is not particularly limited, but is typically in the range of 10 to 50°C, preferably in the range of 15 to 45°C. In addition, it may be batch cleaning or continuous cleaning. The cleaning method using a solid carrier cleaning solution is not particularly limited. Examples include liquid-through cleaning, stirring cleaning, and standing cleaning. Among these, liquid-through cleaning and stirring cleaning are preferred to increase the hydrophilicity of the solid carrier surface, to prevent leakage of protein ligands during isolation, to increase dynamic binding capacity, and to suppress aggregation between carriers. Liquid-through cleaning is more preferred to further improve substitution efficiency, and stirring cleaning is more preferred to suppress aggregation between carriers. A combination of liquid-through cleaning and stirring cleaning is particularly preferred. When performing liquid-through cleaning and stirring cleaning, the order or frequency is not particularly limited, but it is preferable to perform stirring cleaning after liquid-through cleaning and to perform each at least once. When a combination of liquid washing and stirring washing is performed as a solid carrier washing process, the low leakage, low aggregation, and substitution efficiency of proteinaceous ligands are particularly excellent. In addition, when cleaning is performed a total of two or more times, the type of cleaning method used in each time may be the same or different. Liquid-passing cleaning only requires passing a solid carrier cleaning solution through a solid carrier bed one or more times, but when performing liquid-passing cleaning, it is preferable that the solid carrier cleaning process include the following solid carrier bed forming process and solid carrier liquid-passing cleaning process. (Solid Carrier Bed Formation Process) A process of forming a solid carrier bed by filling a solid carrier into a container. (Solid carrier liquid-passing cleaning process) A process of passing the solid carrier bed formed in the solid carrier bed forming process through a solid carrier cleaning solution at least once (preferably at least one cleaning solution selected from a cleaning solution containing hydrogen peroxide, a cleaning solution containing peracetic acid, and a cleaning solution with a pH of 0 or higher and 3 or lower, or a pH greater than 12.5 and 14 or lower (provided that the cleaning solution containing hydrogen peroxide and the cleaning solution containing peracetic acid)). (Solid carrier bed formation process) In the present invention, the "solid carrier bed" refers to a solid carrier solid layer, and may be dry or wet. A specific method for forming the solid carrier bed is to introduce a slurry of solid carriers (e.g., water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, a buffer (carbon dioxide buffer, etc.) as a dispersion medium) into a container, and to settle the solid portion of the solid carriers at the bottom of the container by gravity settling or centrifugal settling, and to remove the liquid phase of the slurry by solid-liquid separation operation (e.g., decantation, centrifugal separation, filtration, etc.). In addition, as a container, a solid-liquid separator is preferred because it is convenient to pass the cleaning liquid directly through the solid carrier bed formed in the solid carrier bed forming process during the solid carrier liquid washing process. Specifically, examples include a column container equipped with a filter, a funnel equipped with a filter, a centrifuge equipped with a filter, and a filter reactor. The content ratio of the solid carrier in the slurry is preferably 5 volume% or more, more preferably 15 volume% or more, and particularly preferably 30 volume% or more, and also preferably 80 volume% or less, more preferably 75 volume% or less, and particularly preferably 70 volume% or less. As for specific ranges, 5 volume% or more and 80 volume% or less in the slurry is preferred, 15 volume% or more and 75 volume% or less is more preferred, and 30 volume% or more and 70 volume% or less is particularly preferred. In addition, the volume percentage of the carrier in the slurry can be calculated by, for example, filling 200 mL of slurry into a glass graduated cylinder with a capacity of 250 mL manufactured by Corning (compliant with JIS R3505 Class A) and letting it stand for 3 hours, and then dividing the settled volume by the volume of the slurry filled into the graduated cylinder. (Solid carrier liquid-passing cleaning process) In the present invention, "washing through liquid" of a solid carrier means passing a cleaning liquid through a solid carrier bed, and cleaning is performed, for example, by passing the cleaning liquid from one direction of the solid carrier bed to the opposite direction. Specifically, cleaning can be performed using a solid-liquid separator such as a filter-equipped column vessel, a filter plate, a filter-equipped funnel, a Buchner funnel, a Nuße filter, a filter-equipped centrifuge, or a filter reactor. Additionally, the cleaning liquid introduced into the solid carrier bed may be incubated preferably for 30 seconds to 3 hours, more preferably for 2 minutes to 60 minutes, and then discharged. In order to increase the hydrophilicity of the solid carrier surface, to make it difficult for protein ligands to leak during isolation, and to suppress aggregation between carriers, the number of times the liquid is passed through the solid carrier is preferably at least once, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 2 to 5 times, and particularly preferably 2 to 3 times. When the number of times the liquid is washed is two or more, the low leakage of protein ligands is particularly excellent. In addition, when the number of times the liquid is washed is three or fewer, the low aggregation is particularly excellent. The type of solid carrier cleaning solution and the amount of solid carrier cleaning solution used per cycle are as described above. In addition, the liquid cleaning may be a liquid cleaning using gravity flow, a cleaning using pressurized liquid, or a liquid cleaning using reduced pressure. (Solid carrier stirring and cleaning process) Stirring wash can be performed by stirring the solid carrier in the solid carrier washing solution, but in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for protein ligands to leak during isolation, to increase the dynamic binding capacity, and to suppress aggregation between carriers, it is preferable to stir wash the solid carrier after the solid carrier washing process at least once in the solid carrier washing solution. In order to increase the hydrophilicity of the solid carrier surface, to make it difficult for protein ligands to leak during isolation, and to suppress aggregation between carriers, the number of stirring washes is preferably 1 or more times, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 1 to 2 times, and particularly preferably 1 time. When the stirring and washing cycle is one or more times, the low coagulation property is particularly excellent. In addition, when the stirring and washing cycle is two or fewer times, the low leakage of protein ligands is particularly excellent. As for the stirring speed of the solid carrier stirring washing process, 10 to 150 rpm is preferred, 20 to 100 rpm is more preferred, and 30 to 80 rpm is particularly preferred. The stirring time per cycle of the solid carrier stirring and washing process is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, even more preferably 2 minutes to 120 minutes, and particularly preferably 5 minutes to 90 minutes. The type of solid carrier cleaning solution and the amount of solid carrier cleaning solution used per cycle are as described above. In addition, when a solid carrier liquid-passing washing process is performed using a solid-liquid separator, continuous stirring washing can be performed by carrying out a solid carrier stirring washing process with the filtrate discharge port of the solid-liquid separator used in the solid carrier liquid-passing washing process open, and batch stirring washing can be performed by using the closed port as a container for the solid carrier stirring washing process. In addition, after the solid carrier washing is finished and prior to the ligand binding process, the washing solution may be removed or the solid carrier may be dispersed into water or a mixture of water and a lower alcohol, if necessary. Here, the ligand binding process is explained. (Ligand binding process) The ligand binding process is a process of binding a proteinaceous ligand to a solid carrier. As a proteinaceous ligand, one or more ligands selected from protein A, protein G, protein L, and derivatives thereof are preferred, and protein A and modified protein A are preferred in order to increase the dynamic binding capacity or to make it difficult for the proteinaceous ligand to leak out during isolation, and modified protein A is more preferred. In addition, protein A includes five domains, E, D, A, B, and C, which have binding ability to immunoglobulin, but among the above ligands, a flexible material of protein A having modified domains of the B and C domains is preferred, and a flexible material of protein A having modified domains of the C domain is more preferred. In addition, among protein ligands, in order to increase dynamic binding capacity or to make it difficult for the protein ligand to leak during isolation, it is preferable to have at least one or two substituted amino acid sequences selected from (a) to (i) below with respect to an amino acid sequence having 85% or more homology with the amino acid sequence represented by SEQ ID NO. 1 (C domain of protein A). Among such protein ligands, it is preferable to have two or more substituted amino acid sequences selected from (a) to (i) below, more preferable to have two to nine substituted amino acid sequences selected from (a) to (i) below, and particularly preferable to have two to six substituted amino acid sequences selected from (a) to (i) below. Furthermore, it is preferable to have two or more such amino acid sequences, more preferable to have two to twelve, and particularly preferable to have four to seven. In the case of containing two or more amino acid sequences, their amino acid sequences may be homologous or heterologous. (a) Substitution of the amino acid residue at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO. 1 with a valine residue (b) Substitution of the amino acid residue at the position corresponding to the 3rd position of the amino acid sequence of SEQ ID NO. 1 with an alanine residue (c) Substitution of the amino acid residue at the position corresponding to the 6th position of the amino acid sequence of SEQ ID NO. 1 with an alanine residue or an aspartate residue (d) Substitution of the amino acid residue at the position corresponding to the 9th position of the amino acid sequence of SEQ ID NO. 1 with an alanine residue (e) Substitution of the amino acid residue at the position corresponding to the 11th position of the amino acid sequence of SEQ ID NO. 1 with an alanine residue, a glutamine residue, or a glutamic acid residue (f) Substitution of the amino acid residue at the position corresponding to position 23 of the amino acid sequence of SEQ ID NO. 1 with a leucine residue (g) Substitution of the amino acid residue at the position corresponding to position 29 of the amino acid sequence of SEQ ID NO. 1 with an alanine residue (h) Substitution of the amino acid residue at the position corresponding to position 43 of the amino acid sequence of SEQ ID NO. 1 with an alanine residue (i) Substitution of the amino acid residue at the position corresponding to position 49 of the amino acid sequence of SEQ ID NO. 1 with an arginine residue As means of substituting amino acid residues, known means such as site-specific mutations of the polynucleotide encoding the domain can be cited. Here, “85% or more homology” regarding the amino acid sequence means preferably 90% or more homology, more preferably 95% or more homology, even more preferably 97% or more homology, even more preferably 98% or more homology, and particularly preferably 99% or more homology. In this specification, the “corresponding position” in the amino acid sequence can be determined by aligning the target sequence and the reference sequence (e.g., the amino acid sequence of SEQ ID NO. 1) to give maximum homology to the conserved amino acid residue present in each amino acid sequence. Alignment can be performed using known algorithms, and the procedure is known to those skilled in the art. For example, alignment can be performed by using the Clustal W multiple alignment program (Thompson, JD et al, 1994, Nucleic Acids Res., 22:4673-4680) in default settings. Clustal W is available, for example, on the website of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) or the Japanese DNA Data Bank (DDBJ [www.ddbj.nig.ac.jp / index.html]) operated by the National Institute of Genetics. In this specification, amino acid residues are also described by the following abbreviations: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). In addition, in this specification, the amino acid sequence of the peptide is described in accordance with the conventional method such that the amino terminus (hereinafter referred to as the N terminus) is located on the left and the carboxyl terminus (hereinafter referred to as the C terminus) is located on the right. In this specification, the "previous" and "postv" positions for a specific position in an amino acid sequence refer to positions adjacent to the N-terminal and C-terminal sides of the said specific position, respectively. For example, when an amino acid residue is inserted at the "previous" and "postv" positions of a specific position, the amino acid residue after insertion is placed at a position adjacent to the N-terminal and C-terminal sides of the said specific position. In a preferred embodiment, the proteinaceous ligand is prepared by performing one or more substitutions selected from (a) to (i) on a proteinaceous ligand (friend domain) having an amino acid sequence having 85% or more homology with the amino acid sequence represented by SEQ ID NO. 1. As a means of substituting a proteinaceous ligand (friend domain) having an amino acid sequence having 85% or more homology with the amino acid sequence represented by SEQ ID NO. 1, a method of introducing a mutation into a polynucleotide encoding the friend domain to cause a substitution of a desired amino acid residue may be used. Specific methods for introducing a mutation into a polynucleotide may include site-specific mutation, homologous recombination, and SOE (splicing by overlap extension)-PCR (Gene, 1989, 77:61-68), and the detailed procedures thereof are well known to those skilled in the art. The prepared ligand has immunoglobulin binding activity and functions as an immunoglobulin binding domain. Preferred examples of proteinaceous ligands include, for an amino acid sequence having 85% or more homology with the amino acid sequence represented by SEQ ID NO. 1 (C domain of protein A), at least two or more substituted amino acid sequences selected from (a) to (i) below are connected in a linear chain. Furthermore, "connected in a linear chain" means a structure in which two or more amino acid sequences are connected in series, either through a linker or without a linker. For example, in the case of being connected through a linker, "connected in a linear chain" means a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are connected in series through a linker, whereas in the case of not being connected through a linker, "connected in a linear chain" means a structure in which the C-terminus of one amino acid sequence and the N-terminus of another amino acid sequence are connected in series by peptide bonds. Specifically, examples include modified protein A which is a 3 to 5 emulsion of an amino acid sequence domain having 85% or more homology with SEQ ID NOs 2 to 4 (provided, it has 85% or more homology with the amino acid sequence represented by SEQ ID NO 1), but in order to make it difficult for the protein ligand to leak out during isolation, it is preferably modified protein A which is a 3 to 5 emulsion of an amino acid sequence domain having 85% or more homology with SEQ ID NOs 2 to 3 (provided, it has 85% or more homology with the amino acid sequence represented by SEQ ID NO 1). The binding amount of the protein ligand is preferably 10 mg or more and 300 mg or less, more preferably 25 mg or more and 150 mg or less, per 1 g of dry weight of the solid carrier to increase the dynamic binding capacity. The binding of a proteinaceous ligand to a solid carrier in the ligand binding process can be carried out in the same manner as conventional methods. As for the ligand binding method, a chemical binding method is preferred. For example, a method of binding a ligand to a functional group capable of binding can be cited. This method can be carried out by referring to the descriptions in pamphlets such as International Publication No. 2015 / 119255 and International Publication No. 2015 / 041218. Specifically, a method of binding an amino group of a ligand to a cyclic ether group, carboxyl group, -C(=O)-OC(=O)-, or formyl group of a solid carrier can be cited. To increase reaction efficiency, the ligand binding reaction is preferably carried out in a buffer with a pH of 7 to 14. In addition, the reaction time of the ligand binding reaction is not particularly limited, but is typically about 0.1 to 72 hours. Furthermore, the reaction temperature can be appropriately selected below the boiling point of the solvent, but is typically about 2 to 100°C. In addition, the ligand may be bound using a method for controlling the orientation of the ligand (U.S. Patent No. 6,399,750, Ljungquist C. et al., rEur.J.Biochem., 1989, Vol. 186, pp. 557-561), a method for binding the ligand to a solid carrier through a linker (spacer) (U.S. Patent No. 5,260,373, Japanese Patent Publication No. 2010-133733, Japanese Patent Publication No. 2010-133734), or a method for accumulating the ligand on a solid carrier by means of an associative group (Japanese Patent Publication No. 2011-256176). As a method for manufacturing a chromatography carrier according to the present invention, in order to increase antifouling properties or to make it difficult for proteinaceous ligands to leak during isolation, a ligand-binding carrier hydrophilization process is further provided between a ligand binding process and a ligand-binding carrier bed formation process, and a ligand-binding carrier containing hydrophilic groups obtained from the ligand-binding carrier hydrophilization process is used as a ligand-binding carrier after the ligand binding process in the ligand-binding carrier bed formation process. (Ligand-binding carrier hydrophilization process) A process of reacting a carrier to which a ligand has been bound via a ligand binding process with a compound having a total of at least two hydrophilic groups within the molecule selected from hydroxyl groups and mercapto groups. As a compound having a total of two or more hydrophilic groups within the molecule used in the hydrophilization process of a ligand-binding carrier, it is preferable to have a compound having a total of two to four hydrophilic groups within the molecule, selected from at least one type of hydrophilic group from hydroxyl groups and mercapto groups, in order to increase antifouling properties or to make it difficult for proteinaceous ligands to leak during isolation. Examples include alcohols having a mercapto group within the molecule, such as mercaptoethanol and thioglycerol; and polyhydric alcohols such as glycerol and diglycerol. Compounds having a total of two or more hydrophilic groups within the molecule may be used as a single type or in combination of two or more types. Among these, alcohols having a mercapto group within the molecule are preferred to enhance antifouling properties or to prevent leakage of proteinaceous ligands during isolation, and thioglycerol is particularly preferred. The total amount of compounds having a total of two or more hydrophilic groups in the molecule in the hydrophilization process of a ligand-bonded carrier is, preferably 1 part by mass or more and 1000 parts by mass or less, more preferably 10 parts by mass or more and 800 parts by mass or less, and particularly preferably 100 parts by mass or more and 600 parts by mass or less, per 100 parts by mass of the ligand-bonded carrier (dry solid content). The hydrophilization process of the ligand-bonded carrier may be carried out in the presence of a basic catalyst. Examples of basic catalysts include triethylamine, N,N-dimethyl-4-aminopyridine, sodium hydroxide, diisopropylethylamine, etc., and one type may be used alone or in combination of two or more types. In addition, the reaction time of the ligand-binding carrier hydrophilization process is not particularly limited, but is typically about 0.5 to 72 hours, and preferably about 0.5 to 48 hours. In addition, the reaction temperature can be appropriately selected below the boiling point of the solvent, but is typically about 2 to 100°C. (Ligand-bound carrier bed formation process, ligand-bound carrier liquid-passing washing process, ligand-bound carrier stirring washing process) The ligand binding carrier bed formation process is a process of forming a ligand binding carrier bed by filling a container with the ligand binding carrier obtained from the ligand binding process. The ligand-binding carrier liquid washing process is a process of washing the ligand-binding carrier bed formed in the ligand-binding carrier bed forming process by passing it through a washing solution (hereinafter also referred to as "ligand-binding carrier washing solution") one or more times. The ligand-binding carrier stirring and washing process is a process of stirring and washing the ligand-binding carrier after the ligand-binding carrier liquid washing process one or more times in the ligand-binding carrier washing solution. The method for manufacturing a chromatography carrier according to the present invention combines and performs a ligand-binding carrier liquid-washing process and a ligand-binding carrier stirring-washing process in this order, thereby not only increasing the dynamic binding capacity of the chromatography carrier for antibodies or their fragments, but also making it difficult for aggregation between carriers to occur and making it difficult for proteinaceous ligands to leak during isolation. Furthermore, the substitution efficiency is also improved. In the present invention, the "ligand-binding carrier bed" refers to a solid layer of a ligand-binding carrier, and may be dry or wet. A specific method for forming a ligand-binding carrier bed may be a method in which a slurry of a ligand-binding carrier (e.g., water, alcohol (ethanol, isopropanol, etc.), a mixture of water and alcohol, a buffer (carbonic acid buffer, etc.) as a dispersion medium) is introduced into a container, and the solid portion of the ligand-binding carrier is settled at the bottom of the container by gravity settling or centrifugal settling, and the liquid phase of the slurry is removed by a solid-liquid separation operation (e.g., decantation, centrifugation, filtration, etc.). In addition, as a container, a solid-liquid separator is preferred because it is convenient to pass the cleaning solution directly through the ligand-binding carrier bed formed in the ligand-binding carrier bed formation process in the ligand-binding carrier bed formation process. Specifically, examples include a column container equipped with a filter, a funnel equipped with a filter, a centrifuge equipped with a filter, and a filter reactor. The content ratio of the ligand-binding carrier in the slurry is preferably 5 volume% or more, more preferably 15 volume% or more, and particularly preferably 30 volume% or more, and also preferably 80 volume% or less, more preferably 75 volume% or less, and particularly preferably 70 volume% or less. As for specific ranges, 5 volume% or more and 80 volume% or less in the slurry is preferred, 15 volume% or more and 75 volume% or less is more preferred, and 30 volume% or more and 70 volume% or less is particularly preferred. In addition, the volume percentage of the carrier in the slurry can be calculated by, for example, filling 200 mL of slurry into a glass graduated cylinder with a capacity of 250 mL manufactured by Corning (compliant with JIS R3505 Class A) and letting it stand for 3 hours, and then dividing the settled volume by the volume of the slurry filled into the graduated cylinder. As a ligand-binding carrier cleaning solution used in the ligand-binding carrier liquid-passing cleaning process and the ligand-binding carrier stirring cleaning process, an aqueous cleaning solution is preferred. "Aqueous cleaning solution" means a cleaning solution containing at least water. Examples of aqueous cleaning solutions include water or a mixture of water and a lower alcohol. Examples of lower alcohols include one or more selected from ethanol and isopropanol. The pH of the ligand-binding carrier washing solution used in the ligand-binding carrier washing process and the ligand-binding carrier stirring washing process is preferably greater than 3, more preferably 6 or higher, even more preferably 7 or higher, even more preferably 8.5 or higher, even more preferably 9.5 or higher, particularly preferably 10.5 or higher, in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for the proteinaceous ligand to leak during isolation, and to increase the dynamic binding capacity; and also, in order to increase the dynamic binding capacity, it is preferably 12.5 or lower, more preferably 12 or lower, and particularly preferably 11.8 or lower. As for specific ranges, a pH greater than 3 and less than or equal to 12.5 is preferred, a pH greater than or equal to 6 and less than or equal to 12.5 is more preferred, a pH greater than or equal to 7 and less than or equal to 12.5 is even more preferred, a pH greater than or equal to 8.5 and less than or equal to 12 is even more preferred, a pH greater than or equal to 9.5 and less than or equal to 12 is even more preferred, and a pH greater than or equal to 10.5 and less than or equal to 11.8 is particularly preferred. According to the method for manufacturing a chromatography carrier of the present invention, a chromatography carrier is obtained in which the proteinaceous ligand is difficult to leak during isolation, even when the pH of the washing solution used in the ligand-binding carrier washing process or the ligand-binding carrier stirring washing process is in a mild pH range as described above. Furthermore, when the pH of the washing solution used in the ligand-binding carrier washing process or the ligand-binding carrier stirring washing process is set to a mild pH range as described above, the dynamic binding capacity increases, thereby achieving both excellent dynamic binding capacity and excellent low leakage. When the pH of the ligand-binding carrier washing solution is set to 8.5 or higher, 9.5 or higher, or 10.5 or higher, the dynamic binding capacity and low leakage of proteinaceous ligands are particularly excellent. In addition, when the pH of the ligand-binding carrier washing solution is set to 12 or lower or 11.8 or lower, the dynamic binding capacity is particularly excellent. It is preferable that the ligand-binding carrier washing solution used in the ligand-binding carrier liquid washing process and the ligand-binding carrier stirring washing process includes a pH adjusting agent to achieve the above pH range. pH adjusters are broadly classified into acidic pH adjusters and basic pH adjusters. Specifically, examples include strong acidic pH adjusters based on inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid; weak acidic pH adjusters based on inorganic acids such as sodium dihydrogen phosphate, carbonate, phosphoric acid, hydrogen fluoride, and hydrogen sulfide; weak acidic pH adjusters based on organic acids such as acetic acid and oxalic acid; strong basic pH adjusters based on inorganic bases such as alkali metal hydroxides, sodium carbonate, and trisodium phosphate; strong basic pH adjusters based on organic bases such as triethylamine; weak basic pH adjusters based on inorganic bases such as sodium bicarbonate, disodium hydrogen phosphate, ammonia, copper hydroxide, magnesium hydroxide, zinc hydroxide, iron hydroxide, and aluminum hydroxide; and weak basic pH adjusters based on organic bases such as diethanolamine. Examples of alkali metal hydroxides include lithium hydroxide, potassium hydroxide, and sodium hydroxide. In addition, the concentration of the pH adjuster is preferably 0.001M or more and 10M or less, more preferably 0.005M or more and 2.5M or less, even more preferably 0.01M or more and 0.5M or less, and particularly preferably 0.05M or more and 0.3M or less in order to make it difficult for the proteinaceous ligand to leak during isolation. When the concentration of the above pH adjuster is 0.05 M or higher, the low leakage of the protein ligand is particularly excellent. In addition, when the concentration of the above pH adjuster is 0.3 M or lower, the dynamic binding capacity is particularly excellent. The ligand-binding carrier washing solution may contain a buffer or two or more pH adjusters to adjust to a desired pH or to maintain a target pH. In the ligand-binding carrier liquid-passing washing process and the ligand-binding carrier stirring washing process, washing using the ligand-binding carrier washing solution may be performed a total of two or more times. However, in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for proteinaceous ligands to leak during isolation, to further improve the substitution efficiency, and to suppress aggregation between carriers, the washing is preferably performed a total of 2 to 20 times, more preferably a total of 2 to 15 times, even more preferably a total of 2 to 10 times, even more preferably a total of 2 to 8 times, and particularly preferably a total of 3 to 4 times. In addition, the type of ligand-binding carrier washing solution used in each step may be of the same type or different type, but the same type is preferred. In addition, the type of washing method used in each step may be of the same type or different type. When the total number of washes using the ligand-binding carrier washing solution is three or more, the substitution efficiency is improved, and the resulting chromatography carrier exhibits particularly excellent low leakage of protein ligands. Additionally, when the total number of washes using the ligand-binding carrier washing solution is four or fewer, the low aggregation property is particularly excellent. The amount of ligand-binding carrier washing solution used per washing step in the ligand-binding carrier liquid-passing washing process and the ligand-binding carrier stirring washing process is preferably 40 parts by volume or more, more preferably 60 parts by volume or more, and particularly preferably 80 parts by volume or more, with respect to 100 parts by volume of dry solids of the ligand-binding carrier, and also preferably 1,000 parts by volume or less, more preferably 500 parts by volume or less, and particularly preferably 300 parts by volume or less, with respect to 100 parts by volume of dry solids of the ligand-binding carrier. As for the specific range, with respect to 100 parts by volume of dry solids of the ligand-binding carrier, it is preferable to be 40 parts by volume or more and 1,000 parts by volume or less, more preferable to be 60 parts by volume or more and 500 parts by volume or less, and particularly preferable to be 80 parts by volume or more and 300 parts by volume or less. The cleaning temperature of the ligand-binding carrier liquid-passing cleaning process and the ligand-binding carrier stirring cleaning process is not particularly limited, but is typically in the range of 10 to 50°C, preferably in the range of 15 to 45°C. In addition, it may be batch cleaning or continuous cleaning. Here, the ligand-binding carrier liquid washing process is explained in detail. In the present invention, "washing through a ligand-binding carrier" means passing a washing solution through a ligand-binding carrier bed, and, for example, washing is performed by passing the washing solution from one direction to the opposite direction of the ligand-binding carrier bed. Specifically, washing can be performed using a solid-liquid separator such as a filter-equipped column vessel, a filter plate, a filter-equipped funnel, a Buchner funnel, a Nutzé filter, a filter-equipped centrifuge, or a filter reactor. Additionally, the washing solution introduced into the ligand-binding carrier bed may be incubated preferably for 30 seconds to 3 hours, more preferably for 2 minutes to 60 minutes, and then discharged. In order to increase the hydrophilicity of the solid carrier surface, to make it difficult for the protein ligand to leak during isolation, to improve the substitution efficiency, and to suppress aggregation between carriers, the number of times the liquid is passed through the ligand-bound carrier liquid is preferably 1 or more times, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 2 to 5 times, and particularly preferably 2 to 3 times. When the number of washes through the liquid is two or more times, the substitution efficiency is improved, and the resulting chromatography support has particularly excellent low leakage of protein ligands. In addition, when the number of washes through the liquid is three or fewer times, the low aggregation is particularly excellent. The total number of times the solid carrier liquid-washing process and the ligand-bound carrier liquid-washing process are preferably 2 or more times, more preferably 2 to 30 times, even more preferably 2 to 16 times, even more preferably 2 to 8 times, even more preferably 3 to 6 times, and particularly preferably 4 to 5 times, in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for proteinaceous ligands to leak during isolation, to improve the substitution efficiency, and to suppress aggregation between carriers. When the total number of times the solid carrier liquid washing process and the ligand-bound carrier liquid washing process are combined is 4 or more, the substitution efficiency is improved, and the resulting chromatography carrier exhibits particularly excellent low leakage of proteinaceous ligands. Additionally, when the total number of times the solid carrier liquid washing process and the ligand-bound carrier liquid washing process are combined is 5 or less, the low aggregation property is particularly excellent. The type of ligand-binding carrier cleaning solution and the amount of ligand-binding carrier cleaning solution used per cycle are as described above. In addition, the liquid cleaning may be a liquid cleaning using gravity flow, a cleaning using pressurized liquid, or a liquid cleaning using reduced pressure. Here, the ligand binding carrier stirring and washing process is explained in detail. In order to increase the hydrophilicity of the solid carrier surface, to make it difficult for the protein ligand to leak during isolation, and to suppress aggregation between carriers, the number of stirring washes is preferably 1 or more times, more preferably 1 to 20 times, even more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 1 to 2 times, and particularly preferably 1 time. When the stirring and washing cycle is one or more times, the low coagulation property is particularly excellent. In addition, when the stirring and washing cycle is two or fewer times, the low leakage of protein ligands is particularly excellent. The total number of stirring washes in the solid carrier stirring washing process and the number of stirring washes in the ligand-bound carrier stirring washing process is preferably 2 times or more, more preferably 2 to 30 times, even more preferably 2 to 16 times, even more preferably 2 to 8 times, even more preferably 2 to 6 times, and particularly preferably 2 to 3 times, in order to increase the hydrophilicity of the solid carrier surface, to make it difficult for proteinaceous ligands to leak during isolation, to improve substitution efficiency, and to suppress aggregation between carriers. As for the stirring speed of the ligand-binding carrier stirring washing process, 10 to 150 rpm is preferred, 20 to 100 rpm is more preferred, and 30 to 80 rpm is particularly preferred. The stirring time per cycle of the ligand binding carrier stirring washing process is preferably 30 seconds to 300 minutes, more preferably 1 minute to 180 minutes, even more preferably 2 minutes to 60 minutes, and particularly preferably 5 minutes to 30 minutes. The type of ligand-binding carrier cleaning solution and the amount of ligand-binding carrier cleaning solution used per cycle are as described above. In addition, when a ligand-binding carrier liquid-passing washing process is performed using a solid-liquid separator, continuous stirring washing can be performed by carrying out a ligand-binding carrier stirring washing process with the filtrate discharge port of the solid-liquid separator used in the ligand-binding carrier liquid-passing washing process open, and batch stirring washing can be performed by using the closed port as a container for the ligand-binding carrier stirring washing process. In addition, the reaction products obtained from each of the above processes may be purified by separation means such as filtration or washing. They may also be classified. Furthermore, according to the method for manufacturing a chromatography carrier of the present invention, it is possible to manufacture a chromatography carrier having a large dynamic binding capacity for an antibody or its fragment, difficulty in leakage of the proteinaceous ligand during isolation, and difficulty in aggregation between carriers. In addition, the substitution efficiency is good and the manufacturing process is simple. In addition, various pH solutions can be used as cleaning solutions for the ligand-binding carrier passing through cleaning process or the ligand-binding carrier stirring cleaning process. For example, even when a cleaning solution with a pH greater than 3 and less than or equal to 12.5 is used, a chromatography carrier is obtained in which the proteinaceous ligand is difficult to leak out during isolation. Furthermore, in this specification, the term "antibody" is a concept that includes any class of immunoglobulins, such as IgG, IgA, IgD, IgE, IgM, and subclasses thereof, and variants thereof. Additionally, in this specification, the term "antibody" may include chimeric antibodies such as humanized antibodies, antibody complexes, and other immunoglobulin modifications comprising an antigen recognition site. In addition, in this specification, "antibody fragment" may be an antibody fragment containing an antigen recognition site or an antibody fragment not containing an antigen recognition site. Examples of antibody fragments not containing an antigen recognition site include proteins containing only the Fc region of immunoglobulin, Fc fusion proteins, and variants or modifications thereof. The volume average particle size of the chromatography carrier obtained as described above is preferably 40 to 150 μm, and more preferably 50 to 100 μm. In addition, the coefficient of variation of the volume average particle size is preferably 40% or less, and more preferably 30% or less. In addition, the specific surface area of ​​the chromatography carrier is preferably 1 to 500 m² / g, and more preferably 10 to 300 m² / g. In addition, the volume average pore diameter of the chromatography carrier is preferably 10 to 300 nm. In addition, the above volume average particle size, coefficient of variation, specific surface area, and volume average pore diameter can be measured by laser diffraction and scattering particle size distribution measurements, etc. In addition, the chromatography carrier obtained as described above is useful for separating antibodies or fragments thereof from a sample containing antibodies or fragments thereof, but the sample may include, for example, blood components such as whole blood, serum, plasma, various blood cells, thrombi, platelets, urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph fluid, bone marrow fluid, tissue fluid, saliva, gastric fluid, joint fluid, pleural fluid, bile, ascites fluid, amniotic fluid, bacterial fluid, cell culture medium, cell culture supernatant, tissue cell lysis fluid, etc. Examples The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. (Preparation of a ligand (immunoglobulin-binding protein)) Immunoglobulin-binding proteins PrA-0 to PrA-3 were obtained. PrA-0 is an immunoglobulin-binding protein comprising a homopentamer in which the C domain of protein A (Sequence No. 1) is linked in series. PrA-1 to PrA-3 are variants in which the mutations described in Table 1 are introduced to each immunoglobulin-binding domain of PrA-0. The expression and purification of PrA-0 to PrA-3 were performed as follows. Escherichia coli BL21(DE3) was transformed using a plasmid encoding PrA-0 to PrA-3, and the obtained transformed cells were cultured in an eutrophic medium at 37°C until the logarithmic growth phase. Afterwards, the target proteins were expressed by adding isopropyl-β-thiogalactopyranoside (manufactured by Wako Junyaku Kogyo Co., Ltd.) at a final concentration of 1 mM to the medium and culturing at 37°C for 4 hours. Subsequently, the culture medium was centrifuged to remove the supernatant, and the cells were lysed by adding a 30 mM Tris buffer at pH 9.5 containing egg white-derived lysozyme (manufactured by Wako Junyaku Kogyo Co., Ltd.) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Junyaku Kogyo Co., Ltd.). From the obtained cell lysate, recombinant immunoglobulin-binding proteins were purified by cation exchange chromatography (SP-Sepharos FF, manufactured by GE Healthcare Biosciences) and anion exchange chromatography (Q-Sepharos FF, manufactured by GE Healthcare Biosciences). The purified immunoglobulin-binding proteins were dialyzed against 10 mM citric acid buffer at pH 6.0. The purity of the recombinant immunoglobulin-binding proteins confirmed by SDS-PAGE was over 95%. [Example 1] (Process 1: Polymerization Process of Porous Particles) 2.69 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water and heated and stirred to dissolve the polyvinyl alcohol, thereby obtaining an aqueous solution. Meanwhile, a monomer composition comprising 3.63 g of divinylbenzene (manufactured by Wako Junyaku Kogyo Co., Ltd.), 0.36 g of 1-ethyl-4-vinylbenzene (manufactured by ChemSampCo.), and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Kosei Co., Ltd.) to prepare a monomer solution. Next, the entire amount of the above aqueous solution was placed into a separable flask, equipped with a thermometer, stirring blades, and a cooling tube, set in a hot water bath, and stirring was started under a nitrogen atmosphere. The entire monomer solution was placed into a separable flask and heated in a hot water bath. When the internal temperature reached 85°C, 1.34 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Junyaku Kogyo Co., Ltd.) was added, and the internal temperature was raised to 86°C. Afterward, stirring was performed for 3 hours while maintaining the temperature at 86°C. Next, the reaction mixture was cooled, filtered, and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water to achieve a particle concentration of 10 mass%, thereby obtaining a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 1". Afterwards, 0.956 g of adipic acid dihydrazide (manufactured by Tokyo Kasei Kogyo Co., Ltd.), 8 g of thioglycerol (manufactured by Tokyo Kasei Kogyo Co., Ltd.), and 1.418 g of diisopropylethylamine (manufactured by Tokyo Kasei Kogyo Co., Ltd.) were added to 100 g of porous particle dispersion 1, heated to 70°C, and stirred for 8 hours while maintaining the temperature at 70°C. Next, the reaction mixture was cooled, filtered, and washed with pure water and ethanol. Next, the particles were dispersed in pure water to achieve a particle concentration of 10 mass%, thereby obtaining a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 2". Next, ethylene glycol diglycidyl ether was reacted with the thioglycerol-derived hydroxyl groups contained in porous particle 2. Specifically, 8.7 g of pure water, 1.2 g of sodium sulfate (manufactured by Wako Junyaku Kogyo Co., Ltd.), and 0.10 g of sodium carbonate (manufactured by Wako Junyaku Kogyo Co., Ltd.) were mixed to obtain a carbon dioxide buffer (pH 11.2). To this carbon dioxide buffer, 0.5 g of ethylene glycol diglycidyl ether (Denacol EX810 manufactured by Nagase Chemtex Co., Ltd.) and 8 mL of porous particle 2 were added, and the mixture was shaken and stirred at 23°C for 16 hours. Next, the reaction mixture was filtered and washed with pure water, and then the particles were dispersed in pure water to achieve a particle concentration of 50 volume% to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particle 3". (Process 2: Cleaning Process of Porous Particles) Next, porous particle 3 was cleaned. That is, a funnel (Kiriyama Seisakusho Kiriyama funnel) lined with a filter (Kiriyama Seisakusho Kiriyama funnel filter paper) was prepared, and 16 mL of a dispersion of porous particles 3 was added to the funnel under reduced pressure. In this way, pure water was filtered from the dispersion by suction filtration, causing the solids of the dispersion to settle at the bottom of the funnel (the bottom of the furnace) to form a bed. After that, 8 mL of a 0.5 M sodium hydroxide aqueous solution (pH 13.7) was added while under reduced pressure suction and passed through, allowing the solution to flow from the top to the bottom of the bed without stirring and filtering as is, a "cut-through washing" (hereinafter also simply referred to as "S" or "washing S") was performed twice. Next, the tip of the funnel was closed, 8 mL of 0.5 M sodium hydroxide aqueous solution (pH 13.7) was added, and the entire slurry in the funnel was stirred (for 10 minutes at 60 rpm) and then suction filtered, and a “re-slurry washing” (hereinafter simply referred to as “R” or “washing R”) was performed once. Next, the particles were dispersed in pure water to a particle concentration of 50 volume% to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 4". (Process 3: Ligand Binding Process) Next, a ligand was bound to porous particle 4. Specifically, 28.8 g of pure water, 5.4 g of sodium sulfate (manufactured by Wako Junyaku Kogyo Co., Ltd.), 0.2 g of sodium bicarbonate (manufactured by Wako Junyaku Kogyo Co., Ltd.), and 0.16 g of sodium carbonate (manufactured by Wako Junyaku Kogyo Co., Ltd.) were mixed to obtain a carbonation buffer (pH 9.3). To 25 mL of this carbonation buffer, 0.17 g of the immunoglobulin-binding protein PrA-1 (modified protein A, which is a pentamer of the amino acid sequence domain of SEQ ID No. 2) prepared in the preparation example and 8 mL of porous particle 4 were added, shaken and stirred at 23°C for 1.5 hours, and the reaction mixture was filtered. Next, a buffer was prepared by mixing 8.8g of pure water, 0.1g of sodium sulfate (manufactured by Wako Junyaku Kogyo Co., Ltd.), and 0.03g of sodium hydroxide (manufactured by Wako Junyaku Kogyo Co., Ltd.), and then adding 4.5g of thioglycerol (manufactured by Tokyo Kasei Kogyo Co., Ltd.). The hydrophilization reaction solution was added to porous particles bound to ligands, and the hydrophilization reaction was carried out by shaking and stirring at 23°C for 16 hours. Next, the particles were dispersed in pure water so that the particle concentration was 50 volume%, thereby obtaining a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous particles 5". (Process 4: Cleaning process after the joining process) Next, porous particle 5 was cleaned. That is, a funnel (Kiriyama funnel manufactured by Kiriyama Seisakusho) lined with a filter (filter paper for Kiriyama funnel manufactured by Kiriyama Seisakusho) was prepared, and 16 mL of a dispersion of porous particles 5 was added to the funnel under reduced pressure. In this way, by filtering and separating pure water from the dispersion by suction filtration, the solids of the dispersion settled at the bottom of the funnel (the bottom of the furnace) to form a bed. Afterwards, "washing S" was performed twice by adding 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4) while under vacuum suction and passing it through. Next, the tip of the funnel was closed, 8 mL of 0.1 M sodium carbonate aqueous solution (pH 11.4) was added, and the entire slurry in the funnel was stirred (for 10 minutes at 60 rpm), and then a "washing R" was performed once by suction filtration. Next, after neutralizing with sodium citrate buffer, the particles were dispersed in pure water to a particle concentration of 50 volume% to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "carrier 1". [Example 2] Carrier 2 was obtained by performing the same operation as in Example 1, except that the 0.5M aqueous sodium hydroxide solution used in "Process 2: Cleaning Process of Porous Particles" was changed to 0.1M hydrochloric acid. [Example 3] Carrier 3 was obtained by performing the same operation as in Example 1, except that the 0.5M aqueous sodium hydroxide solution used in "Process 2: Cleaning Process of Porous Particles" was changed to 1.0M hydrochloric acid. [Example 4] Carrier 4 was obtained by performing the same operation as in Example 1, except that the 0.5M aqueous sodium hydroxide solution used in "Process 2: Cleaning Process of Porous Particles" was changed to 0.01M hydrochloric acid. [Example 5] Carrier 5 was obtained by performing the same operation as in Example 1, except that the 0.5M sodium hydroxide aqueous solution used in "Process 2: Cleaning Process of Porous Particles" was changed to a 0.1M hydrogen peroxide aqueous solution. [Example 6] Carrier 6 was obtained by performing the same operation as in Example 1, except that the 0.5M sodium hydroxide aqueous solution used in "Process 2: Cleaning Process of Porous Particles" was changed to a 0.1M peracetic acid aqueous solution. [Example 7] Carrier 7 was obtained by performing the same operation as in Example 1, except that the 0.5M sodium hydroxide aqueous solution used in "Process 2: Cleaning Process of Porous Particles" was changed to a 1.0M sodium hydroxide aqueous solution. [Example 8] Carrier 8 was obtained by performing the same operation as in Example 1, except that the 0.5M sodium hydroxide aqueous solution used in "Process 2: Cleaning Process of Porous Particles" was changed to a 0.1M sodium hydroxide aqueous solution. [Example 9] A carrier 9 was obtained by performing the same operation as in Example 1, except that the "cleaning S" performed in "Process 2: Cleaning Process of Porous Particles" was changed from 2 times to 4 times. [Example 10] A carrier 10 was obtained by performing the same operation as in Example 1, except that the “cleaning S” performed in “Process 2: Cleaning Process of Porous Particles” was changed from 2 times to 1 time. [Example 11] A carrier 11 was obtained by performing the same operation as in Example 1, except that the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Process 4: Cleaning Process After Bonding Process" was changed to a 0.1M sodium phosphate aqueous solution (prepared by mixing a 0.1M sodium dihydrogen phosphate aqueous solution and a 0.1M disodium hydrogen phosphate aqueous solution to achieve a pH of 7.5). [Example 12] A carrier 12 was obtained by performing the same operation as in Example 1, except that the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Process 4: Cleaning Process After Bonding Process" was changed to a 0.5M sodium phosphate aqueous solution (prepared by mixing a 0.5M sodium dihydrogen phosphate aqueous solution and a 0.5M disodium hydrogen phosphate aqueous solution to achieve a pH of 7.5). [Example 13] A carrier 13 was obtained by performing the same operation as in Example 1, except that the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Process 4: Cleaning Process After Bonding Process" was changed to a 0.01M sodium phosphate aqueous solution (prepared by mixing a 0.01M sodium dihydrogen phosphate aqueous solution and a 0.01M sodium hydrogen phosphate aqueous solution to achieve a pH of 7.5). [Example 14] A carrier 14 was obtained by performing the same operation as in Example 1, except that the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Process 4: Cleaning Process After Bonding Process" was changed to a 0.1M sodium carbonate aqueous solution (prepared by adding sodium hydroxide aqueous solution to achieve a pH of 12.5). [Example 15] A carrier 15 was obtained by performing the same operation as in Example 1, except that the 0.1M sodium carbonate aqueous solution (pH 11.4) used in "Process 4: Cleaning Process After Bonding Process" was changed to a 0.1M sodium carbonate aqueous solution (prepared by mixing a 0.1M sodium bicarbonate aqueous solution and a 0.1M sodium carbonate aqueous solution to achieve a pH of 10). [Example 16] A carrier 16 was obtained by performing the same operation as in Example 1, except that the “cleaning S” performed in “process 4 cleaning process after bonding process” was changed from 2 times to 4 times. [Example 17] A carrier 17 was obtained by performing the same operation as in Example 1, except that the “cleaning S” performed in “Process 4 cleaning process after bonding process” was changed from 2 times to 1 time. [Example 18] A carrier 18 was obtained by performing the same operation as in Example 1, except that the porous particle 3 obtained in "Process 1: Polymerization Process of Porous Particles" was changed to an agarose-based particle (WorkBeads 40 ACT (manufactured by Bio-works)). [Example 19] A carrier 19 was obtained by performing the same operation as in Example 1, except that the immunoglobulin binding protein PrA-1 used in "Process 3: Ligand Binding Process" was changed to the immunoglobulin binding protein PrA-0. [Example 20] A carrier 20 was obtained by performing the same operation as in Example 1, except that the immunoglobulin binding protein PrA-1 used in "Process 3: Ligand Binding Process" was changed to the immunoglobulin binding protein PrA-3. [Example 21] A carrier 21 was obtained by performing the same operation as in Example 1, except that the immunoglobulin binding protein PrA-1 used in "Process 3: Ligand Binding Process" was changed to the immunoglobulin binding protein PrA-2. [Example 22] A carrier 22 was obtained by performing the same operation as in Example 1, except that the immunoglobulin binding protein PrA-1 used in "Process 3: Ligand Binding Process" was changed to Pierce (registered trademark) Recombinant Protein L (Thermo Fisher Scientific No. 21189). [Example 23] A carrier 23 was obtained by performing the same operation as in Example 1, except that the immunoglobulin binding protein PrA-1 used in "Process 3: Ligand Binding Process" was changed to Pierce (registered trademark) Recombinant Protein G (Thermo Fisher Scientific 21193). [Comparative Example 1] A carrier of Comparative Example 1 was obtained by performing the same operation as in Example 1, except that the “cleaning S” performed in “Process 4 cleaning process after bonding process” was changed from 2 times to 3 times and the “cleaning R” was changed from 1 time to 0 times. [Comparative Example 2] A carrier of Comparative Example 2 was obtained by performing the same operation as in Example 1, except that the “cleaning S” performed in the “cleaning process after the 4th bonding process” was changed from 2 times to 0 times and the “cleaning R” was changed from 1 time to 3 times. [Comparative Example 3] A carrier of Comparative Example 3 was obtained by performing the same operations as in Example 1, except that “Process 2: Cleaning process of porous particles” was not performed, the 0.1M sodium carbonate aqueous solution (pH 11.4) used in “Process 4: Cleaning process after bonding process” was changed to a 0.5M sodium hydroxide aqueous solution (pH 13.7), the “Cleaning S” performed in “Process 4: Cleaning process after bonding process” was changed from 2 times to 6 times, and the “Cleaning R” was changed from 1 time to 0 times. [Comparative Example 4] A carrier of Comparative Example 4 was obtained by performing the same operations as in Example 1, except that the “Process 2 cleaning process of porous particles” was not performed, the 0.1M sodium carbonate aqueous solution (pH 11.4) used in the “Process 4 cleaning process after bonding process” was changed to a 0.5M sodium hydroxide aqueous solution (pH 13.7), the “cleaning S” performed in the “Process 4 cleaning process after bonding process” was changed from 2 times to 0 times, and the “cleaning R” was changed from 1 time to 3 times. (Test Example 1) Dynamic coupling capacity (DBC) measurement test Using the AKTA avant25 manufactured by Cytiva, the DBC of each carrier in Examples 1 to 21 and Comparative Example was measured for a protein (human IgG antibody, manufactured by LGC 1875-0007) at a retention time of 4 minutes. A column vessel with a capacity of 4 mL (5 mmφ × 200 mm length) was used, and the protein was prepared by dissolving 5 mg / mL of protein in a 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5). The DBC was calculated from the protein capture amount at the elution front 10% breakthrough and the column packing volume, and evaluated according to the following criteria. The results are shown in Tables 2 to 4 and 6. (DBC evaluation criteria) AA (Excellent): 62 mg / mL or higher A (Good): 61 mg / mL or more and less than 62 mg / mL B (Poor): Less than 61 mg / mL (Test Example 2) Protein Leakage Measurement Test Using the AKTA avant25 manufactured by Cytiva, 7.5 mL of cell culture medium (Herceptin, titer: 4.38 mg / ml) was loaded onto each carrier of the example and comparative example at a retention time of 4 minutes, and then the antibody was recovered as the eluent. A column vessel with a capacity of 0.8 mL (5 mmφ × 40 mm length) and a 100 mM aqueous sodium acetate solution (pH 3.3) were used as the eluent, and washing with a 20 mM sodium phosphate / 500 mM sodium chloride solution (pH 7.5) was performed before elution. Next, the Protein L-Ligand Leakage ELISA Kit (manufactured by Genaxxon Bioscience) was used for the carrier of Example 22, the Protein G ELISA Kit (manufactured by Alpha Diagnostic International) was used for the carrier of Example 23, and the Protein A ELISA kit (F740) was used for other carriers to determine the amount of protein in the eluent and the antibody concentration in the eluent from the absorbance, respectively. From these values, the amount of protein leak per amount of antibody in the recovered eluent was calculated and evaluated according to the following criteria. The results are shown in Tables 2 to 6.

[0131] (Evaluation criteria for protein leakage amount) AAA (Excellent): 8 ppm / IgG or less AA (Excellent): Greater than 8 ppm / IgG and less than 10 ppm / IgG A (Good): 10 ppm / IgG or higher, less than 13 ppm / IgG B (Poor): 13 ppm / IgG or higher (Test Example 3) Evaluation of Coagulation Amount 16 mL of dispersion of each carrier of the Examples and Comparative Examples was passed through a metal mesh (manufactured by Taiyo Co., Ltd., mesh-replaceable funnel mesh 30 mesh), and the particles remaining on the mesh were recovered using pure water. The recovered particle dispersion was transferred to an aluminum dish and heated on a hot plate at 200°C for 10 minutes to obtain dried particles. The weight of these dried particles was measured and recorded as the aggregation amount (g). The smaller the value of the obtained aggregation amount, the less aggregation is considered to be. The results are shown in Tables 2 to 6. (Test Example 4) Replacement efficiency of the cleaning solution The electrical conductivity of the cleaning solution initially used in "Process 4: Cleaning Process After the Bonding Process" was measured using an electrical conductivity meter manufactured by HORIBA. Subsequently, the final filtrate immediately before cleaning with sodium citrate buffer in "Process 4: Cleaning Process After the Bonding Process" was recovered, and its electrical conductivity was measured in the same manner as above. The replacement efficiency (%) of the cleaning solution was calculated using the following formula. The closer this value is to 100%, the better the replacement efficiency is considered to be. The results are shown in Tables 2 to 6. Replacement efficiency of the cleaning solution (%) = (Electric conductivity of the filtrate / Electric conductivity of the cleaning solution) × 100 (Test Example 5) Evaluation of relative hydrophilicity 30 parts by mass of water was added to 10 parts by mass of dry porous particles 4 (particles before ligand binding) obtained in processes 1 to 2 of each example and comparative example, and the dispersion state was visually confirmed. "A" was determined as particles that were neatly dispersed without clumping, "B" as particles that were dispersed but showed small clumps, and "C" as particles that had large clumps remaining. The results are shown in Tables 2 to 6.

Claims

Claim 1 A method for manufacturing a chromatography carrier comprising the following steps: (Ligand binding process), a ligand binding carrier bed formation process, a ligand binding carrier liquid washing process, and a ligand binding carrier stirring washing process. (Ligand binding process) A process of binding a proteinaceous ligand to a solid carrier (Ligand binding carrier bed formation process); a process of filling a ligand binding carrier obtained in the ligand binding process into a container to form a ligand binding carrier bed (Ligand binding carrier liquid washing process); a process of washing the ligand binding carrier bed formed in the ligand binding carrier bed formation process with a washing solution one or more times (Ligand binding carrier stirring washing process); a process of stirring and washing the ligand binding carrier after the ligand binding carrier liquid washing process one or more times in the washing solution. Claim 2 A method for manufacturing a chromatography carrier according to claim 1, wherein the number of times the ligand binding carrier is washed through the liquid washing process is 2 to 5. Claim 3 A method for manufacturing a chromatography carrier according to claim 1 or 2, further comprising a solid carrier washing process, wherein the solid carrier washing process comprises the following solid carrier bed forming process and solid carrier liquid washing process, and the solid carrier washed in the solid carrier washing process is used as the solid carrier in a ligand binding process. (Solid carrier bed forming process) A process of filling a solid carrier into a container to form a solid carrier bed. (Solid carrier liquid washing process) A process of washing the solid carrier bed formed in the solid carrier bed forming process by passing it through a washing solution one or more times. Claim 4 A method for manufacturing a chromatography carrier according to any one of claims 1 to 3, further comprising a solid carrier washing process, wherein the solid carrier washing process comprises the following solid carrier bed forming process, solid carrier liquid washing process, and solid carrier stirring washing process, and wherein the solid carrier washed in the solid carrier washing process is used as the solid carrier in a ligand binding process. (Solid carrier bed forming process) A process of filling a solid carrier into a container to form a solid carrier bed. (Solid carrier liquid washing process) A process of washing the solid carrier bed formed in the solid carrier bed forming process by passing it through a washing solution one or more times. (Solid carrier stirring washing process) A process of washing the solid carrier after the solid carrier liquid washing process by stirring it in a washing solution one or more times. Claim 5 A method for manufacturing a chromatography carrier according to claim 3 or 4, wherein the number of times the solid carrier is washed through the liquid washing process is 2 to 5 times. Claim 6 A method for manufacturing a chromatography carrier according to any one of claims 3 to 5, wherein the total number of times the solid carrier liquid washing process and the ligand-binding carrier liquid washing process are combined is 2 to 8 times. Claim 7 A method for preparing a chromatography carrier according to any one of claims 1 to 6, wherein the proteinaceous ligand is one or more ligands selected from protein A, protein G, protein L and materials thereof.

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